2.1. Field of the Invention
This invention, in the fields of biochemistry and cell and molecular biology, relates to a novel protein tyrosine phosphatase (PTP) termed PTP 1D. Included is the PTP 1D protein, nucleic acid constructs coding therefor, recombinant expression vectors comprising the nucleic acid construct, cells containing or expressing the recombinant expression vectors, methods for producing and identifying PTP 1D protein and DNA constructs, antibodies specific for PTP 1D protein and glycoprotein, and methods for screening compounds capable of binding to the inhibiting or stimulating protein tyrosine phosphatase enzymatic activity of PTP 1D.
2.2. Description of the Background Art
2.2.1. Introduction
Phosphorylation of proteins is a fundamental mechanism for regulating diverse cellular processes. While the majority of protein phosphorylation occurs at serine and threonine residues, phosphorylation at tyrosine residues has attracted much interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase (PTKase or PTK) activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Hunter et al., Ann. Rev. Biochem. 54:987-930 (1985); Ullrich et al., Cell 61:203-212 (1990); Nurse, Nature 344:503-508 (1990); Cantley et al., Cell 64:281-302 (1991)).
The phosphorylation of protein tyrosine residues is a dynamic process with competing phosphorylation and dephosphorylation reactions. These processes are regulated by the reciprocal actions of PTKs, which catalyze tyrosine phosphorylation, and protein tyrosine phosphatases (PTPases or PTPs), which specifically dephosphorylate tyrosine residues of phosphorylated proteins. The net level of tyrosine phosphorylation of intracellular proteins is thus determined by the balance of PTK and PTP enzymatic activities. (Hunter, T., Cell 58:1013-1016 (1989)).
2.2.2. Protein Tyrosin Kinases
PTKs comprise a large family of proteins, including many growth factor receptors and potential oncogenes which share ancestry with, but nevertheless differ from, serine/threonine-specific protein kinases (Hanks et al., Science 241:42-52 (1988)). Many PTKs have been linked to initial signals in the induction of the cell cycle (Weaver et al., Mol. Cell. Biol. 11:4415-4422 (1991)).
Most of our current understanding of mechanism underlying changes in PTKs comes from receptor-type PTKs (RPTKs) having a transmembrane topology. The binding of a specific ligand to the extracellular domain of an RPTK is thought to induce oligomerization, increasing the enzymatic (kinase) activity and activation of the signal transduction pathways (Ullrich et al., supra). Dysregulation of kinase activity through mutation or overexpression is a well xe2x80x94xe2x80x94established mechanism underlying cell transformation (Hunter et al., 1985supra; Ullrich et al., supra).
2.2.3. Protein Tyrosine Phosphatases
The protein phosphatases comprise at least two separate and distinct families (Hunter, T., 1989, supra): protein serine/threonine phosphatases and protein tyrosine phosphatases (PTPs). The PTPs are themselves a family, containing at least two subgroups. The first subgroup comprises low molecular weight, intracellular enzymes that contain a single conserved catalytic phosphatase domain. Members of this subgroup include:
(1) placental PTP 1B (Charbonneau et al., Proc. Natl. Acad. Sci. USA 86:5252-5256 (1989); Chernoff et al., Proc. Natl. Acad. Sci. USA 87:2735-2789 (1989));
(2) T-cell PTP (Cool et al., Proc. Natl. Acad. Sci. USA 86:5257-5261 (1989));
(3) rat brain PTP (Guan et al., Proc. Natl. Acad. Sci. USA 87:5201-1502 (1990));
(4) neuronal phosphatase (STEP) (Lomborso et al., Proc. Natl. Acad. Sci. USA 88:7242-7246 (1991)); and
(5) cytoplasmic phosphatases that contain a region of homology to cytoskeletal proteins (Gu et al., Proc Natl. Acad. Sci. USA 88:5867-57871 (1991); Yang et al., Proc. Natl. Acad. Sci. USA 88:5949-5953 (1991)).
Since the first PTP was purified, sequenced and cloned, additional potential PTPs have been identified at a rapid pace, and the number continues to grow steadily. The large number of known members of the PTP family suggests that there may be specificity in PTP-RPTK interactions. A cDNA encoding a novel PTP designated PTP 1C was cloned form several sources (Shen, S. -H. et al.,i Nature 352:736-739 (1991); Plutzky, J. et al., Proc. Natl. Acad. Sci. USA 89:1123xe2x80x94(1992); Yi, T., et al., Mol. Cell Biol. 12:836-846 (1992 ); Matthews, R. J. et al., Molec. Cell. Biol. 12:2396xe2x80x94(1992)). The PTP 1C protein has a single catalytic domain and a pair of N-terminally located src-homology regions, termed SH2, suggesting that PTK activity could be directly regulated by SH2 domain-mediated interaction with a PTP.
The second PTP subgroup includes the high molecular weight, receptor-linked PTPs, termed RPTPs. RPTPs consist of (a) an intracellular catalytic region, (b) a single transmembrane segment, an (c) a putative ligand-binding extracellular domain (Gebbink, M. F. et al., FEBS Lett. 290:123-130 (1991)). The structures and sizes of the putative xe2x80x9cextracellular receptorxe2x80x9d domains of various RPTPs are diverse, whereas the intracellular catalytic domains are highly conserved. All RPTPs have two tandemly duplicated catalytic phosphatase homology domains, with the exception of HPTPO, which has only one. Tsai et al., J. Biol. Chem. 266:10534-10543 (1991)).
One RPTP, originally named the leukocyte common antigen (LCA) (Ralph, S. J., EMBO J. 6:1251-1257 (1987)), has been known by other names, including T200 (Trowbridge et al., Eur. J. Immunol. 6:557-562 (1962 )), B220 for the B cell form (Coffman et al., Nature 289:681-683 (1981)), the mouse allotypic marker Ly-5 (Komuro et al., Immunogenetics 1:452-456 (1975)), and more recently, CD45 (Cobbold et al., Leucocyte Typing III, McMichael et al., eds., pp. 788-803, 1987). The LCA molecules comprise a family of high molecular weight glycoproteins expressed on the surface of all leukocytes and their hemopoietic progenitors (Thomas, Ann. Rev. Immunol. 7:339-369 (1989)), and have remarkable sequence homology between animal species (Charbonneau et al., Proc. Natl. Acad. Sci. USA 85:7182-7186 (1988)). CD45 is thought to play a critical role in T cell activation. (For review, see: Weiss A., Ann. Rev. Genet. 25:487-510 (1991).) Thus, mutagenized T cell clones which did not express CD45 were functionally impaired in responding to stimulation via the T cell receptor (Weaver et al., 1991, supra). CD45 PTP activity played a role in the activation of pp56kk, a lymphocyte-specific PTK (Mustelin et al., Proc. Natl. Acad. Sci. USA 86:6302-6306 (1989); Ostergaard et al., Proc. Natl. Acad. Sci. USA 86:8959-8963 (1989)). These findings led to the hypothesis that T cell activation involved the phosphatase enzyme activating pp56kk by dephosphorylation of a C-terminal tyrosine residue.
Another RPTP, the leukocyte common antigen related molecule, LAR (Streuli et al., J. Exp. Med. 168:1523-1530 (1988)), was initially identified as an LCA homologue in which the intracellular catalytic region had two catalytic phosphatase homology domains (domains I and II). However, only domain I appeared to have phosphatase activity (Streuli et al., EMBO J. 9(8):2399-2407 (1990)). Chemically-induced LAR mutants (tyr1379xe2x86x92phe) were temperature-sensitive (Tsai et al., J. Biol. Chem. 266(16):10534-10543 (1991)).
A murine RPTP, designated mRPTPxcexc, has an extracellular domain sharing structural motifs with LAR (Gebbink et al., supra). The human homologue of RPTPxcexc was cloned, and the gene was localized to human chromosome 18. Two Drosophila PTPs, termed DLAR and DPTP were predicted based on the sequences of cDNA clones (Streuli et al., Proc. Natl. Acad. Sci. USA 86:8698-8702 (1989)). cDNA encoding another Drosophila RPTP, DPTP 99A, has also been cloned and characterized (Hariharan et al., Proc. Natl. Acad. Sci. USA 88:11266-11270 (1991)).
Other examples of RPTPs include RPTP-xcex1, xcex2, "Ugr", and xcex6 (Krueger et al., EMBO J. 9:3241-3252 (1990), Sap et al., Proc. Natl. Acad. Sci. USA 87::6112-6116 (1990), Kaplan et al., Proc. Natl. Acad. Sci. USA 87:7000-7004 (1990), Jirik et al., FEBS Lett. 273:239-242 (1990), Mathews et al., Proc. Natl. Acad. Sci. USA 87:4444-4448 (1990), Ohagi et al., Nucl. Acids Res. 18:7159 (1990)). PCT Publication WO92/01050 discloses human RPTP-xcex1, xcex2 and xcex8, and the nature of the structural homologies found among the conserved domains of these three RPTPs and other members of this protein family. An intracellular domain of murine RPTP-xcex1 is homologous to the catalytic domains of other PTPs. The 142 amino acid extracellular domain (including signal peptide) of RPTP-xcex1has a high serine and threonine content (32%) and 8 potential N-glycosylation sites. cDNA clones encoding RPTP-xcex1 have been produced and expressed in eukaryotic hosts. Natural expression of RPTP-xcex1 protein in various cells and tissues was detected with a polyclonal antibody to RPTP-xcex1, produced by immunization with a synthetic RPTP-xcex1 peptide. This antibody detected a 130 kDa protein in cells transfected with a cDNA clone encoding a portion of RPTP-xcex1.
Another RPTP, HEPTP, was discovered by screening of a hepatoblastoma cell line (HepG2) cDNA library with a probe encoding the two PTP domains of LCA (Jirik et al., FASEB J. 4A:2082, Abstr 2253 (1990)). The HEPTP gene appeared to be expressed in a variety of human and murine cell lines and tissues.
The PTP D subfamily of PTPs was disclosed in a commonly assigned, related U.S. Patent application Ser. No. 07/923,740, filed Aug. 5, 1992, the entire contents of which are hereby incorporated by reference.
Conserved amino acid sequences in the catalytic domains of known PTPs have been identified (Krueger et al., EMBO J. 9:324-3252 (1990); Yi et al., Mol. Cell. Biol. 12:836-846 (1992), both of which references are incorporated herein by reference in their entirety). These amino acid sequences are designated xe2x80x9cconsensus sequencesxe2x80x9d herein. Yi et al. aligned the catalytic phosphatase domain sequences of LCA, PTPIB, TCPTP, LAR, DLAR, HPTPxcex1, HPTPxcex2 and HPTPxcex8, identifying the following xe2x80x9cconsensus sequencesxe2x80x9d (See: Yi et al., supra, FIG. 2(A), lines 1-2):
1. D Y I N A S/N [SEQ. ID NO: 1]
2. K C X X Y W P [SEQ. ID NO. 2]
Krueger et al., aligned the catalytic phosphatase domain sequences of PTP1B, TCPTP, LAR, LCA, HPTPxcex1, HPTPxcex8, HPTPxcex5, and HPTPxcex6, DLAR and DPTP, identifying the following xe2x80x9cconsensus sequences xe2x80x9d (See: Krueger et al., supra, FIG. 7, lines 1-2):
1. D/N Y I N A S/N [SEQ. ID NO. 3]
2. K C X X Y W P [SEQ. ID NO. 2]
Inclusion of the PTP 1D, csw (corkscrew) and PTP 1C in the sequence comparisons revealed that the conserved sequences QGP is altered in the SH2 domain-containing phosphatases to QGC.
Dephosphorylation of tyrosine residues can, by itself, function as an important cellular regulatory mechanism. Thus, with the src family of tyrosine kinasis, dephosphorylation of a C-terminal tyrosine activated the kinase enzymatic activity (Hunter, T., Cell 49:1-4 (1987)). Tyrosine dephosphorylation may be an obligatory step in the mitotic activation of the maturation-promoting factor (MPF) kinase (Morla et al., Cell 58:193-203 (1989)).
2.2.4 Interactions between Protein Tyrosine Kinases and Phosphatases
Cellular factors involved in signalling include polypeptide substrates which contain the src-homologous regions designated SH2 and SH3, either alone or in combination with an enzymatic activity (Koch, C. A. et al., Science 252:668 (1991); Russell, R. B. et al., FEBS Lett. 304:15 (1992)) For example, phospholipase Cxcex8 is activated upon interaction with and phosphorylation by the cytoplasmic domain of a RPTK (Margolis, B. et al., Cell 57:1101-1107 (1989); Meisenshelder, J. et al., Cell 57:1109 (1989); Burgess, W. H. et al., Mol. Cell. Biol. 10:4470 (1990); Nishibe, S. et l., Science 250:1253 (1990)). While PTPs are thought to be regulators of PTKs, the activation of these crucial components of phosphotyrosine signalling cascades are still not understood (Fischer, E. H. et al., Science 253:401 (1991); Pot, D. A. et al., Biochim. Biophys. Acta 1136:35 (1992)).
The existence of a large number of PTP family members (described above) suggest that there may be specificity in interactions between particular PTPs and PTKs. The structure of the PTP 1C molecule, discussed above, includes, in addition to a single catalytic domain, a pair of N-terminally-located SH2 regions. The presence of these SH2 regions suggests that PTK activity can be directly regulated by SH2 domain-mediated interaction with a PTP.
The above observations point out the need in the art for understanding the mechanisms that regulate PTP activity. Further analysis of structure-function relationships among PTPs are needed to gain important understanding of the mechanisms of signal transduction, cell cycle progression and cell growth, neoplastic transformation and the fundamental changes in a number of important diseases including cancer and diabetes.
The inventors describe herein the identification, cloning and sequencing of a novel protein tyrosine phosphatase (PTP) designated PTP 1D which differs significantly in structure from most previously reported PTPs, and has 71% sequence similarity with PTP 1C.
Thus, the present invention provides a PTP 1D protein, or a functional derivative thereof, wherein, when the PTP 1D protein is one which occurs in nature, the PTP 1D protein is substantially free of other proteins or glycoproteins with which it is natively associated.
Preferably, the PTP 1D protein comprises an amino acid sequence that has 71% or more identity with the amino acid sequence of PTP 1D shown in FIG. 2 (SEQ ID NO:5]. More preferably, the PTP 1D protein comprises SEQ ID NO:5.
A substantially pure PTP 1D protein, may be produced by biochemical purification, or may be prepared by chemical means or by recombinant means in a prokaryotic or eukaryotic host, and is provided substantially free of other proteins with which it is natively associated and/or has modified amino acids.
The functional derivative of the PTP 1D protein, includes a fragment, a protein or peptide with additional or substituted amino acids, a PTP 1D protein, having any combination of deleted, additional, or substituted amino acids, such that the PTP 1D protein possesses the desired biological activity.
Also provided herein is a recombinant nucleic acid construct comprising a nucleotide sequence encoding a PTP 1D protein, or encoding a functional derivative thereof. Such nucleic acid construct may be a cDNA or a genomic DNA molecule. It is preferably an expression vehicle such as a plasmid. In a preferred embodiment, the nucleic acid construct encodes PTP 1D and comprises the nucleotide sequence shown in FIG. 2 [SEQ ID NO:4].
Also provided is an isolated and purified nucleic acid construct comprising a nucleotide sequence encoding the PTP 1D protein. Preferably, the nucleotide sequence is SEQ. ID NO:4.
The present invention includes as a prokaryotic and a eukaryotic host cell transformed or transfected with, or otherwise containing, the above expression vehicle or plasmid.
Also provided is a method for preparing a PTP 1D protein, comprising:
(a) culturing a host cell capable of expressing the PTP 1D protein under culturing conditions,
(b) expressing the PTP 1D protein; and
(c) recovering the PTP 1D protein from the culture.
The present invention is also directed to an antibody, including a polyclonal, monoclonal, or chimeric antibody, specific for an epitope of a PTP 1D protein.
The invention is further directed to a method for detecting the presence of or measuring the quantity of PTP 1D in a cell, the method comprising:
(a) contacting the cell or an extract thereof with an antibody specific for an epitope of PTP 1D protein; and
(b) detecting the binding of the antibody to the cell or extract thereof, or measuring the quantity of antibody bound, thereby determining the presence of or measuring the quantity of the PTP 1D protein.
Also provided is a method for detecting in a nucleic acid containing sample the presence of a nucleic acid sequence encoding a normal or mutant PTP 1D protein, comprising:
(a) contacting the sample, or an extract thereof, with an oligonucleotide probe encoding at least a portion of the normal or mutant PTP 1D protein under hybridizing conditions; and
(b) measuring the hybridization of the probe to nucleic acid of the sample, thereby detecting the presence of the nucleic acid sequence.
Preferably, the above method includes, before step
(a), the step of selectively amplifying the amount of nucleic acid encoding the at least a portion of the normal or mutant PTP 1D protein.
The present invention is further directed to a method for identifying a compound capable of binding to PTP 1D protein, the method comprising:
(a) attaching the PTP 1D protein, or a compound-binding portion thereof, to a solid phase matrix;
(b) contacting a sample suspected of containing the compound with the matrix-bound PTP 1D protein, glycoprotein or portion thereof, allowing the compound to bind, and washing away any unbound material; and
(c) detecting the presence of the compound bound to the solid phase matrix.
In another embodiment is provided a method for isolating from a complex mixture a compound capable of binding to PTP 1D protein, the method comprising:
(a) attaching the PTP 1D protein, or a compound binding portion thereof, to a solid phase matrix;
(b) contacting the complex mixture with the matrix-bound PTP 1D protein, glycoprotein or portion thereof, allowing the compound to bind, and washing away any unbound material; and
(c) eluting the bound compound from the solid phase matrix, thereby isolating the compound.
In another embodiment, the present invention includes a method for determining whether a compound is capable of stimulating or inhibiting the phosphotyrosine phosphatase enzymatic activity of PTP 1D, the method comprising:
(a) contacting the compound with the PTP 1D protein in pure form, in a membrane preparation, or in a whole live or fixed cell, or with an enzymatically active fragment of the PTP 1D protein;
(b) incubating the mixture of step (a) for an interval sufficient for the compound to stimulate or inhibit the enzymatic activity;
(c) measuring the phosphotyrosine phosphatase enzymatic activity of the PTP 1D protein, glycoprotein or fragment;
(d) comparing the enzymatic activity to that of the PTP 1D protein, glycoprotein or fragment incubated without the compound, thereby determining whether the compound stimulates or inhibits the activity.
The present invention is also directed to a pharmaceutical composition for treating or preventing a disease associated with an abnormal PTP 1D protein, the composition comprising a PTP 1D protein, or a functional derivative thereof, and a pharmaceutically acceptable carrier.
In another embodiment, the invention provides a pharmaceutical composition for treating or preventing a disease associated with an abnormal PTP 1D protein having deficient enzymatic activity, the composition comprising a compound in an amount effective to stimulate phosphotyrosine phosphatase enzymatic activity of PTP 1D, and a pharmaceutically acceptable carrier.
Also provided is a pharmaceutical composition for treating or preventing a disease associated with an abnormal PTP 1D protein having supranormal enzymatic activity, the composition comprising a compound in an amount effective to inhibit phosphotyrosine phosphatase enzymatic activity of PTP 1D, and a pharmaceutically acceptable carrier.
The present invention includes a method for treating or preventing a disease associated with an abnormal PTP 1D protein in a subject, comprising administering to the subject of pharmaceutical composition as described above.